Process for producing pravastatin sodium salt using streptomyces flavidovirens dsm 14455

ABSTRACT

In one aspect, the present invention provides an improved method for the manufacture of Pravastatin sodium salt by fermentation under optimal fermentation parameters using a new strain of  Streptomyces flavidovirens.

PRIORITY CLAIM

The present application claims the benefit under 35 U.S.C. § 371 ofInternational Application No.: PCT/IN01/00161, filed Sep. 27, 2001, theentire contents of which is hereby incorporated herein by reference.

FIELD OF THE INVENTION

In one aspect, the present invention relates to a process for themanufacture and purification of Pravastatin sodium salt using a newmicroorganism, Streptomyces flavidovirens BICC 6826.

BACKGROUND OF THE INVENTION

Lovastatin, pravastatin, compactin, and derivatives and analogs thereof,are known to be potent HMG-CoA reductase inhibitors and are used asantihyper-cholesterolemic agents. Lovastatin, compactin and pravastatinare produced by fermentation, using microorganisms of different speciesbelonging to Aspergillus, Penicilium and Streptomyces genera,respectively.

The purity of the active ingredient is an important factor formanufacturing a safe and effective pharmaceutical, especially if thepharmaceutical product must be taken on a long-term basis in thetreatment or prevention of high plasma cholesterol. Accumulation ofimpurities from pharmaceuticals of lower purity may cause many sideeffects during medical treatment of a subject.

Among all the statins produced by microorganisms, pravastatin is hassignificant advantages in that it exhibits stronger and highlytissue-selective inhibition of cholesterol synthesis (Tsujita et. al,Biochim Biophy Acta, 1986, 877, 50–60). Pravastatin is produced bymicrobial hydroxylation of its precursor, Compactin (also calledML-236B). This bioconversion is carried out by a number ofmicroorganisms e.g. Streptomyces (U.S. Pat. No. 5,179,013, U.S. Pat. No.4,448,979), Nocardia, Amycolata, Saccharopolyspora, Amycolatopsis,Saccharothrix, Gilbertella (EP 0649907, WO 99/60151), Actinomadura (WO96/40863), Mortierella (WO 00/46175), Nocardia (U.S. Pat. No. 5,830,695)and Bacillus sp. (U.S. Pat. No. 6,245,535, WO 99/07827). A number ofspecies of Streptomyces, e.g. S. carbophilus, S. hastedii (JP4,349,034), S. flavovirens (WO 99/10419), S. rosenchromogenous (U.S.Pat. No. 4,346,227), S. californicus (EP 649907) and S. exfoliatus (WO98/45410) are also known to carry out this bioconversion.

The bioconversion is a cytochrome p450 dependent system and the enzymesare induced by the presence of compactin in the medium (Matsuoka et al,European Journal of Biochemistry, 1989: 184: 707–713; Serizawa et. al,In: Biotechnology of antibiotics; W R Strohl (editor) 1997). Compactinmust often be added into the seed medium for efficient bioconversion (WO98/45410). Attempts have been made to clone and express this system byrecombinant DNA techniques in a fungal host (e.g. Penicillium citrinum)for one step de novo production of pravastatin (WO 99/10499). However,the yields are not economically viable. Bioconversion using Streptomycesspecies is still currently the most efficient method of pravastatinproduction.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a new strain Streptomycesflavidovirens BICC 6826. In another aspect, there is provided a processfor efficient conversion and purification of compactin, a compactin saltor a compactin derivative, to pravastatin sodium salt using a new strainof Streptomyces.

DETAILED DESCRIPTION OF CERTAIN PREFERRED EMBODIMENTS OF THE INVENTION

In certain embodiments, the present invention provides a process for themanufacture and purification of Pravastatin sodium salt, comprisingsteps of:

-   (i) preparing a seed inoculum of a strain of Streptomyces    flavidovirens capable to 6β-hydroxylating a compound of formula II:

-   -   wherein R is an alkali metal or ammonium ion,

-   (ii) transferring the seed inoculum to a production medium,

-   (iii) subjecting the production medium to fermentation,

-   (iv) feeding a substrate of Formula II in the production medium at    different intervals,

-   (v) controlling the pH during fermentation by feeding a carbon    source in the production medium,

-   (vi) fermenting the substrate until bioconversion to Pravastatin is    complete,

-   (vii) extracting Pravastatin from the whole cell broth, and

-   (viii) isolating Pravastatin sodium salt of formula I

In certain embodiments, the strain of Streptomyces flavidovirens isStreptomyces flavidovirens BICC6826.

In certain other embodiments, the strain of Streptomyces flavidovirensis Streptomyces flavidovirens strain deposited at DSMZ (DeutscheSammlung von Mikro-organismen und Zellkulturen GmbH) under accessionnumber DSMZ 14455.

In yet other embodiments, the inoculum used for the seed is a sporesuspension or a vegetative mycelium.

In still other embodiments, the seed medium comprises malt extract andpeptone.

In certain other embodiments, the pH of the seed medium is between about6.0 and 7.5 before sterilization.

In yet other embodiments, the seed medium is incubated at between about25 and 35° C. for about 40 to 55 hours.

In still other embodiments, constituents of the production medium areselected from a group consisting of dextrose monohydrate, peptone andyeast extract.

In certain other embodiments, the production medium has a pH of betweenabout 6.0 and 7.5 before sterilization.

In yet other embodiments, the production medium is incubated betweenabout 24 and 35° C. for about 48 to 148 hours.

In still other embodiments, the substrate used for feeding is compactin,a compactin salt or a compactin derivative.

In certain other embodiments, the pH is controlled by feeding a carbonsource selected from a saccharide or glycerol.

In addition to providing a new strain of Streptomyces flavidovirens BICC6826, the present invention has several advantages over the otherreported methods, including using a new strain of Streptomycesflavidovirens BICC 6826; resuting in a higher bioconversion rate,therefore making the process economically attractive; and involvingfewer steps for the isolation and purification process.

Streptomyces has been the most commonly used microorganism as itscytochrome system has been well studied (EP 281245, U.S. Pat. No.5,830,695). Among the Streptomyces, S. flavidovirens offers a uniqueadvantage for bioconversion. Unlike S. exfoilatus (WO 98/45410) S.flavidovirens does not need induction during its vegetative phase. Incertain embodiments, bioconversion is between 40 and 90%, preferablybetween 60 and 80%; with compactin concentrations in the vegetativebroth between 0.05 and 10 g/L, preferably between 3 and 6 g/L.

Unlike many cultures reported in the art where the bioconversion is slowand is carried out over 12 days of fermentation (e.g. Mortierellamaculata, WO 00/46175), S. flavidovirens carries out conversion within24 hrs to 7 days, preferably between 2 to 5 days.

The illustrated embodiments have been set forth only for the purpose ofexample and should not be taken as limiting the invention. Therefore, itshould be understood that within the scope of the appended claims, theinvention may be practiced other than specifically described herein.

EXEMPLIFICATION Example 1 Seed Inoculum Preparation

About 100 μL spore suspension of Streptomyces flavidovirens BICC 6826,made by adding 3 mL of sterile water to a culture slant, was added to a250 mL Erlenmeyer flask containing 35 mL of medium containing 30 g/Lmalt extract and 5 g/L peptone. pH was adjusted to 6.8 beforesterilization. The seed flasks were incubated at 28° C. on a rotaryshaker (200-rpm) for 48 hours.

Example 2 Seed Inoculum Preparation

Seed inoculum was prepared in the same way as in Example 1 but the sporesuspension was replaced by 1 mL of vegetative mycelium stored inglycerol.

Example 3 Bioconversion to Pravastatin

In 250 mL Erlenmeyer flasks containing 35 mL of production mediumcontaining 20 g/L Dextrose monohydrate, 10 g/L Peptone and 1 g/L yeastextract, was added about 0.5 mL of seed inoculum from Example 2. Beforeinoculation, the pH of the medium was adjusted to 7.0 and the flaskswere sterilized for 30 min at 121° C.

The flasks were then incubated on a rotary shaker (200-rpm) at 28° C.After 2 days of incubation, a sterile solution of compactin sodium saltsodium was added along with sterile dextrose feed (50% w/v). Thebioconversion was estimated after 24 hrs by harvesting one of themultiple flasks running under similar conditions. This procedure wasrepeated every 24 hrs until 3 mg/mL of compactin was fed cumulatively.The maximum amount of pravastatin accumulated at the end of experimentwas about 1.5 mg/mL.

Example 4 Bioconversion to Pravastatin

Bioconversion was carried out in 250 mL Erlenmeyer flasks in the sameway as described in Bioconversion 3 but 7 mg/mL of dextrose feed wasadded every 24 hours along with compactin solution. About 2.0 mg/mL ofpravastatin was detected in the flasks after 120 hrs of incubation.

Example 5

Bioconversion to Pravastatin

Bioconversion was carried out in a 2L-stirred tank bioreactor with 1.7 Lmedium. In addition to the medium components described in Example 3,0.1% (v/v) of silicone antifoam was added before autoclaving the medium.

Seed inoculum (3.5%) was transferred aseptically to the bioreactor andthe culture was allowed to grow at 28° C. The dissolved oxygenconcentration was maintained above 25% of saturation. After 48 hrs ofincubation, about 1 mg/ml of compactin feed was added each day. Dextrosefeed was also added along with every compactin feed. The pH of thereaction mixture was maintained between 7.6 to 8.0 by adding dextrosefeed as needed. After 4 days, 1.6 g/L of pravastatin was produced.

Example 6 Bioconversion to Pravastatin

Bioconversion was carried out in a similar way as in Example 5, but nosugar was added along with the compactin feed.

About 0.5 mg/ml of compactin feed was added in every compactin addition.The pH of the reaction mixture was maintained between 8.4 and 8.6 byadding dextrose feed as needed. About 46% of the 2.9 g/L of compactinfed to the fermentation vessel was detected as pravastatin.

Example 7 Bioconversion to Pravastatin

Bioconversion was carried out as described in Example 3, but thecompactin was added as an ammonium salt. 0.9 g of the total compactinfed (2.9 g/L) was assayed as pravastatin.

Example 8 Extraction of Broth

After fermentation, the whole cell broth was obtained and the pH wasadjusted to 12 and held for 1 hr. 90% of the total product present wasextracted into the supernatant.

Example 9 Scale up Studies on Hydrophobic Interaction Resin

A hydrophobic interaction resin, SP825, was packed in a XK 26/70(Pharmacia) column. The pH 12 extracted and filtered broth from Example8 was passed through a pre-equilibrated column and pravastatin wasbound. A washing step was done using pH 12 water. Elution was done withmethanol. The overall recovery from the broth extract was 92%.

Example 10 Separation of Pravastatin Using Secondary Amine

80 ml of methanolic extract from the HIC resin containing 10 gpravastatin sodium salt was acidified to pH 4 using dilute HCl and thepravastatin in the acid form was extracted into equal volume of ethylacetate.

120 Mole % of a dibenzylamine was added to the ethyl acetate extract,and the resulting mixture was stirred for half-hour and then chilled at4° C. for 1–2 hours. The resulting pravastatin dibenzyamine salt wasseparated by filtration. The crystals were washed with ethyl acetate,filtered and dried. A pravastatin bibenzylamine salt containing 9 gequivalent of pravastatin acid was obtained.

Example 11 Precipitation of Sodium Salt of Pravastatin Using SodiumCaprylate

Around 5 g of pravastatin amine salt was dried and dissolved in 20 ml of10% NaOH solution and washed with ethyl acetate. The pH was adjusted to4 using dilute hydrochloric acid. The resulting mixture was furtherextracted into equal volume of ethyl acetate.

The ethyl acetate layer was washed with brine, and activated carbon wasadded and stirred for 30 min at room temperature. The resulting mixturewas filtered and washed with ethyl acetate. The ethyl acetate solutionwas dried over anhydrous sodium sulfate.

To the above ethyl acetate solution was added 1.2 gms of sodiumcaprylate, and the resulting solution was stirred for 2 hrs at roomtemperature. 12.5 ml of acetonitrile was added and the resultingsolution was stirred for 1 hr. The reaction mixture was cooled to 5° C.,and stirred for 1 hr.

The pravastatin sodium salt precipitate was filtered and washed withchilled acetonitrile. The compound was dried under vacuum to givepravastatin sodium in yield of 90% and an assay purity of greater than99%.

Example 12 Purification of Pravastatin

The broth (10000 L) was acidified to pH=4 by adding 50% ortho phosphoricacid and an equal volume of ethyl acetate was added. The layers wereseparated and the organic layer was washed with water, concentratedunder reduced pressure to give a total volume of about 300 L, andstirred under reflux for 24 h. The solution was cooled to roomtemperature, washed with 5% sodium bicarbonate solution, followed bywater, and concentrated under reduced pressure to 125 L. The residue waschilled to 0° C. and stirred for 2 h. The solid obtained was filtered togive the lactone.

To 1 Kg of the lactone obtained from the above step, methanol (1 L) and10% sodium hydroxide (2 L) were added and the resulting mixture wasstirred for 0.5 h at room temperature. Water (1 L) and ethyl acetate (2L) were added and the contents were stirred for 10 min at roomtemperature. The ethyl acetate layer was separated and discarded. The pHof the aqueous layer was carefully adjusted to 4 using 6N HCl and wasextracted with ethyl acetate (4 L). The layers were separated, and theethyl acetate layer washed with brine. Activated charcoal was added. Theresulting mixture was filtered and the filtrate was dried over sodiumsulfate. After filtration, sodium caprylate (347 g) was added to thefiltrate and the contents were stirred for 1 h, after which timeacetonitrile (2.5 L) was added. Stirring was continued for additional 2h, the solution was chilled to 0° C., and the solid pravastatin sodiumsalt that precipitated was filtered and dried at 40° C. under vacuum.

The crude sodium salt was dissolved in 2 L of water and acetonitrile wasadded (30 L) over a period of 2 h. The contents were chilled to 0° C.and were stirred for 4 h at 0° C. The resulting solid was filtered andis washed with acetonitrile. The solid was dried under vacuum at 40° C.to give pharmaceutical grade pravastatin sodium salt.

Example 13 Purification of Pravastatin

To the ethyl acetate layer obtained in Example 12, sodium acetate wasadded instead of sodium caprylate, and the resulting mixture wasprocessed to give pharmaceutical grade pravastatin sodium salt.

Example 14 Purification of Pravastatin

100 gm crude pravastatin lactone, obtained in a similar way as inExample 12 was added to 300 ml alkaline methanolic solution. The pH ofthe solution was adjusted to 4 and the pravastatin acid was extractedinto 400 ml Ethyl acetate. 120 Mole % dibenzylamine was added to theethyl acetate solution. The resulting solution was stirred for 1 hour,chilled at 4° C. and filtered. The crystals were dissolved in methanol,the pH was adjusted to 4 by addition of dilute hydrochloric acid andpravastatin was extracted into ethyl acetate. To the ethyl acetatesolution was added 90 mole % sodium caprylate, and the resulting mixturewas stirred well. Crystals of the sodium salt precipitated out whenacetonitrile was added to the solution, which was chilled for 1 hour.The product was obtained in 70% yield with greater than 99% assaypurity.

1. A strain of Strepomyces flavidovirens deposited at Deutsche Sammlungvon Mikro-organismen und Zelikulturen GmbH under accession number DSM14455.
 2. A microbial process for preparing a compound of formula I

from a substrate compound of general formula II,

wherein R represents an alkali metal or ammonium ion, comprising stepsof (i) preparing a seed inoculum of a strain of Streptomycesflavidovirens that 6β-hydroxylates a compound of formula II, (ii)transferring the seed inoculum to a production medium, (iii) subjectingthe production medium to fermentation, (iv) feeding a substrate ofFormula II in the production medium at different intervals, (v)controlling the pH during fermentation by feeding a carbon source in theproduction medium, (vi) fermenting the substrate until bioconversion toPravastatin is complete, (vii) extracting Pravastatin from theproduction medium, and (viii) isolating Pravastatin sodium salt.
 3. Theprocess of claim 2, wherein the strain of Streptomyces flavidovirens isthe Strepbomyces flavidovirens strain deposited with DSMZ (DeutscheSammlung von Mikro-organismen und Zellkulturen GmbH) under accessionnumber DSMZ
 14455. 4. The process of claim 2 wherein the inoculum usedfor the seed is a spore suspension or a vegetative mycelium.
 5. Theprocess of claim 2 wherein the substrate is compactin, a compactin saltor a compactin derivative.
 6. The process of claim 2 wherein the seedinoculum comprises malt extract and peptone.
 7. The process of claim 2further comprising a step of incubating the seed inoculum.
 8. Theprocess of claim 7 wherein the seed medium is incubated between about 25and 35° C. for about 40 to 55 hours.
 9. The process of claim 2 whereinconstituents of the production medium are selected from the groupconsisting of dextrose monohydrate, peptone and yeast extract.
 10. Theprocess of claim 2 further comprising a step of incubating theproduction medium.
 11. The process of claim 10 wherein the productionmedium is incubated between about 24 and 35° C. for about 48 to 148hours.
 12. The process of claim 2 wherein the carbon source is selectedfrom a saccharide or glycerol.
 13. The process of claim 12 wherein thecarbon source is dextrose.
 14. The process of claim 2 wherein the stepof isolating Pravastatin sodium salt comprises converting Pravastatinisolated from the cell broth to its dibenzylamide salt.
 15. The processof claim 14 wherein the dibenzylamide salt is converted to Pravastatinsodium salt by reaction with sodium caprylate.
 16. The process of claim2 wherein in the step of extracting Pravastatin from the whole cellbroth, Pravastatin is obtained in its lactone form.
 17. The process ofclaim 2 wherein in the step of extracting Pravastatin from the wholecell broth, Pravastatin is obtained as its lactone form, and the step ofisolating Pravastatin sodium salt comprises converting the lactone toPravastatin acid.
 18. The process of claim 17 wherein converting thelactone to Pravastatin acid comprises contacting the lactone with amethanolic solution of sodium hydroxide.
 19. The process of claim 17wherein Pravastatin acid is further converted to Pravastatin sodium saltby reaction with sodium caprylate.
 20. The process of claim 17 whereinPravastatin acid is further converted to Pravastatin sodium salt byreaction with sodium acetate.
 21. The process of claim 17 whereinPravastatin acid is further converted to Pravastatin dibenzylamide saltby reaction with dibenzylamine.
 22. The process of claim 21 whereinPravastatin dibenzylamide salt is further converted to Pravastatinsodium salt by reaction with sodium caprylate.
 23. A compositioncomprising a strain Streptomyces flavidovirens DSM 14455 and compactin,a compactin salt or a compactin derivative.